Practically all life forms require iron. Most iron in the environment is oxidized and virtually insoluble. A further complication is that iron's aqueous chemistry can generate highly toxic oxidizing species. Hence organisms must tightly regulate both the quantity and the state of iron in the cell. In humans, the failure to acquire adequate iron from the diet results in iron deficiency anemia, a major nutritional problem worldwide. In addition, imbalances in iron metabolism such as hereditary hyperferritinemia and hemochromatosis can lead to a number of diseases in humans, including blindness, cirrhosis, cardiac disease, neurological disorders, and cancer. Iron transport, utilization, and storage in animal cells is regulated post-transcriptionally by two iron regulatory proteins, IRP1 and IRP2. The IRPs reversibly bind to highly conserved stem-loop structures known as iron responsive elements (IREs) in the mRNAs encoding the iron transport and storage proteins, thereby providing coordinated, reciprocal iron regulation. The IRPs are themselves regulated by the cellular iron status, where high intracellular iron concentrations suppress their IRE binding activities. High iron levels convert IRP1 into a cytosolic aconitase enzyme, complete with an Fe-S cluster. Thus IRP1 is bifunctional, and the interconversion between its two forms underlies its regulation of gene expression. The IRP: IRE system is one of the best-understood post-transcriptional mechanisms of regulation, but until now, no structural information was available. We have recently determined the structure of three IRP1: IRE complexes. Using those results, we have developed a model to explain IRPI's selectivity in multiple-IRE recognition, and we have also proposed a model for the switch mechanism that regulates IRP1 interconversion. To test these hypotheses, and to complete the long-term objective of explaining the molecular details of control by this key metabolic regulator, we will 1) Determine the structural basis for IRE recognition and differential regulation by IRP1;and 2) Determine the nature of the switch mechanism in IRP1. These experiments will be performed using the methods of molecular biology and x-ray crystallography. The results will potentially generate novel targets for therapeutic approaches to diseases of iron metabolism.